Identification of FOXO1 as a geroprotector in human synovium through single-nucleus transcriptomic profiling

Abstract The synovium, a thin layer of tissue that is adjacent to the joints and secretes synovial fluid, undergoes changes in aging that contribute to intense shoulder pain and other joint diseases. However, the mechanism underlying human synovial aging remains poorly characterized. Here, we generated a comprehensive transcriptomic profile of synovial cells present in the subacromial synovium from young and aged individuals. By delineating aging-related transcriptomic changes across different cell types and their associated regulatory networks, we identified two subsets of mesenchymal stromal cells (MSCs) in human synovium, which are lining and sublining MSCs, and found that angiogenesis and fibrosis-associated genes were upregulated whereas genes associated with cell adhesion and cartilage development were downregulated in aged MSCs. Moreover, the specific cell-cell communications in aged synovium mirrors that of aging-related inflammation and tissue remodeling, including vascular hyperplasia and tissue fibrosis. In particular, we identified forkhead box O1 (FOXO1) as one of the major regulons for aging differentially expressed genes (DEGs) in synovial MSCs, and validated its downregulation in both lining and sublining MSC populations of the aged synovium. In human FOXO1-depleted MSCs derived from human embryonic stem cells, we recapitulated the senescent phenotype observed in the subacromial synovium of aged donors. These data indicate an important role of FOXO1 in the regulation of human synovial aging. Overall, our study improves our understanding of synovial aging during joint degeneration, thereby informing the development of novel intervention strategies aimed at rejuvenating the aged joint.


Supplemental materials
Figure S1.Aging-related histological changes in human subacromial synovium (A) IHC staining of HP1γ in young and aged human synovial tissues.Left, representative images.Scale bars, 50 μm and 10 μm (zoomed-in images).Right, the intensity of HP1γ in the indicated region (L or SL) is quantified as fold changes (Aged vs. Young).n = 6 individuals per group.
(B) Immunofluorescence staining of CD45 in young and aged human synovial tissues.Left, representative images.Scale bars, 50 μm and 20 μm (zoomed-in images).Yellow arrows indicate CD45 positive cells of zoom-in images.Right, the number of CD45 positive cells per field of vision in SL region is quantified as fold changes (Aged vs. Young).n = 6 individuals per group.
Statistical significance was assessed using two-tailed Student's unpaired t test.Data are presented as the mean ± SEM.P values less than 0.05 were considered statistically significant.(B) Western blot analysis of the FOXO1 in FOXO1 +/+ and FOXO1 -/-hESCs.GAPDH was used as the loading control.
Statistical significance was assessed using two-tailed Student's unpaired t test (F).Data are presented as the mean ± SEM. (F) Scatter plots showing the expression levels of FOXO1 correlated genes and FOXO1 based on the snRNA-seq data.In both young and aged groups, every 20 nuclei of L-MSC and SL-MSC were aggregated into a single point.The gene expression levels for each point were calculated as the average expression of the nuclei within that specific point.
(G) Table showing the NES values and motifDb data of SDK1, which is an overlapping gene between the FOXO1 correlated genes and the FOXO1 target genes predicted by SCENIC in L-MSC and SL-MSC.MotifDb data indicated the predicted binding sites within 500 bp upstream of the transcription start site (TSS) for the target gene.
Statistical significance was assessed using one-way ANOVAs with Dunnett's multiple comparison tests (C) or two-tailed Student's unpaired t test (D).Data are presented as the mean ± SEM. ns, not significant, **P < 0.01, ***P < 0.001.

Supplementary Table Legends
Table S1.Sample information used in this study.Table S2.Marker genes of different cell types identified in snRNA-seq of human synovium.Table S3.Aging-related differentially expressed genes of bulk RNA-seq and snRNA-seq datasets.
Table S4.Core regulatory transcription factors of differentially expressed genes in human synovium during aging.
Table S5.List of primers and probes used in this study.
Table S6.Source of indicated gene sets.

Figure S2 .
Figure S2.Aging-related transcriptional changes in human synovium (A) Bar plots showing the number of cells (left) and the percentage of reads mapped to genome (right) in each group.(B) Box plots showing the number of genes detected (left) and the number of unique molecular identifiers (right) in each cell across different groups.(C) UMAP plot showing the average expression of classical marker genes across different cell types in the human synovium.Color key indicates the expression level of marker genes in each cell.(D) Ridge map showing the global distribution density of gene set score of fibrosis-related genes of young and aged human synovial tissues.The corresponding dashed line represents the median position of each group.(E) Ridge map showing the global distribution density of gene set score of SASP of young and aged human synovial tissues.(F) Violin plot showing the gene set score of SASP across different cell types of young and aged human synovial tissues.(G) GSEA enrichment curves show the change of the aging-related pathway by bulk RNA-seq.

Figure S3 .
Figure S3.Changes in cell-cell interaction pairs during human synovial aging (A) Bubble plot showing the representative enriched functional annotations for decreased cell-cell interaction pairs during synovial aging.(B) Dot plot showing the high-frequency (frequency ≥ 5) aged-specific cell-cell interaction pairs in indicated cell types across young and aged groups.The size of the dots represents the -Log10P value and the color key indicates the mean value of expression levels.(C) Dot plot showing the high-frequency (frequency ≥ 4) young-specific cell-cell interaction pairs in indicated cell types across young and aged groups.The size of the dots represents the -Log10P value and the color key indicates the mean value of expression levels.

Figure S4 .
Figure S4.Generation and characterization of FOXO1 -/-hESCs (A) Schematic diagram of FOXO1 gene editing strategy using TALEN mediated homologous recombination gene editing in hESCs.